Our overall goal is to understand how biological systems that contain paramagnetic metal centers and free radicals work. We will probe the paramagnetic centers to determine local liganding structure, shared unpaired electron distribution, and evidence for protein-induced perturbation. We will investigate these centers primarily through the techniques of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR). Where mechanism needs to be clarified, EPR- related stopped-flow and freeze-quench methods will be employed. Our la is one of the few in the world which routinely uses ENDOR to measure hyperfine coupling of electron spins in biological molecules. The systems to be studied are: 1. Manganese Centers. At the Mn2+ center in pyruvate kinase we will closely examine the presently uncertain binding mode of phosphoenolpyruvate substrate and pyruvate product. Then in study aimed at the multimanganese photosynthetic oxygen-evolving center and models for it we will probe by ENDOR for evidence of exchangeable oxygen bound to that center. 2. Ribonucleotide Reductase. We will perform ENDOR measurements to uncover the structure of early radical intermediates in the assembly of the diiron/tyrosyl radical cofactor that is needed for nucleotide reduction. 3. Activated Bleomycin. We will examine the presently unknown mode of oxygen-iron ligation in the activated bleomycin that is a necessary precursor to bleomycin-induced DNA damage. 4. Paramagnetic Centers of Electron Transport. With the goal of understanding the heme's liganding environment, we will probe the heme- copper catalytic core of terminal cytochrome c and quinol oxidases where oxygen is reduced to water. At specialized ubiquinone binding sites of the electron transport chain we will investigate the structure and kinetic behavior of stabilized semiquinone radical. 5. Nitrite Reductase. By using native and genetically altered forms of this bacterial enzyme, we will clarify the active site structure of its Type-1 and Type-2 coppers that serve to convert nitrite to nitric oxide. With EPR monitoring we will measure redox behavior of these metal centers, and with freeze-quench trapping we will explore the enzymatic mechanism.